Solid carriers with peptide nucleic acid probes and methods for regeneration

ABSTRACT

A method for the detection of point mutation and polymorphisms in nucleic acids or for sequencing of unknown nucleic acids by a simple procedure using arrays uses nucleic acid analoges as sequence discriminators. This procedure simplifies the working mode in complex problematic cases.

This is a Division of application Ser. No. 08/894,808 filed Nov. 12,1997, now U.S. Pat. No. 6,475,721, issued Nov. 5, 2002. The disclosureof the prior application is hereby incorporated by reference herein inits entirety.

Subject matter of the invention is a solid carrier having two or morenucleic acid analogs with different base sequences bound topredetermined sites on its surface. The invention also addresses amethod for the detection of nucleic acids using a carrier of thisnature.

Sample analysis has undergone rapid development in recent decades. Whileanalytes were initially detected primarily by means of their reactionwith conventional chemical reagents, and later on with enzymes, teststhat utilize the immunological characteristics of the analyte havebecome the standard recently, especially in medical diagnostics This isespecially true in the field of infectious diseases. However,immunological procedures can basically only detect analytes with whichimmunologically active compounds such as antigens or antibodies play arole. These procedures have resulted in promising potential applicationsfor many infections caused by viruses or bacteria. Genetic diseases orpredispositions that are not expressed as a change in proteinpatterns—or only to an insufficient extent—are either difficult orimpossible to detect using immunological procedures, however. Nucleicacids have therefore recently become the object of detection in manycases. The presence of certain nucleic acids can infer the presence ofan infectious agent or the genetic condition of an organism. Detectionprocedures based on the presence of special nucleotide sequences inparticular were facilitated recently when methods for the amplificationof nucleic acids that are present in small numbers became available. Dueto the large quantity of sequence information and the fact that twonucleotide sequences with completely different functions often differ byjust one base unit, the specific detection of nucleotide sequences stillposes a considerable challenge for reagents and analytical methods thatare based on the detection of nucleic acid sequences. In addition, thenucleotide sequences are often not even known, but rather are determinedfor the first time in the nucleic acid detection method itself.

A method for the detection of nucleotide sequences of the HLA gene isdescribed in EP-B-0 237 362 with which a clinically relevant pointmutation can be detected. In this method, an oligonucleotide that isbound to a membrane and has a nucleotide sequence that is exactlycomplementary to one of the two nucleic acids to be differentiated isbrought in contact with the sample. While certain conditions aremaintained, only that nucleic acid that is exactly complementary bindsto the oligonucleotide that is bound to the solid phase, and can bedetected.

A method is described in Proc. Natl. Acad. Sci. USA 86, 6230–6234 (1989)in which a large number of oligonucleotides that are bound to different,predetermined sites of a nylon membrane by means of poly-dT are used forthe simultaneous detection of all known allelic variants of an amplifiedregion of a nucleic acid.

A method is described in U.S. Pat. No. 5,202,231 in which the sequenceof a nucleic acid can be determined theoretically by bringingoligonucleotides having a predetermined, known sequence in contact witha sample of the unknown nucleic acids under hybridization conditions.This requires that all possible permutations of the nucleotide sequencebe immobilized on known sites of a solid phase. By determining the sitesto which the nucleic acids containing the sequence to be determinedhybridize, it can theoretically be determined which sequences arepresent in the nucleic acid.

Prior art in the field of the analysis of genetic polymorphisms using“oligonucleotide arrays” is described in Nucleic Acids Research 22,5456–5465 (1994) and Clin. Chem. 41/5, 700–6 (1995).

The main problem with the prior art is the fact that the meltingtemperatures of the selected sequence-specific oligonucleotidescontaining the nucleic acids to be sequenced or detected are different.To remedy this situation, one has to perform the complex method ofselecting the length of the oligonucleotide and its base composition,and optimizing the position of the mismatches within the oligonucleotideas well as the salt concentration of the hybridization complex. In manycases, however, it is practically impossible to simultaneouslydistinguish closely related sequences from each other. The hybridizationtemperature is another critical parameter. Variations of as little as 1to 2° C. can change the intensity or produce false-negative results.Incorrect analytical results based on the presence of point mutationshave serious implications for diagnosis.

The object of this invention was, therefore, to provide an alternativemethod for the sequence-specific detection of nucleic acids and toprovide suitable materials for this method.

This object was accomplished by providing a solid carrier having two ormore nucleic acid analogs with different base sequences bound topredetermined sites on its surface. Another object of the invention is amethod for the sequence-specific detection of a nucleic acid using thissolid carrier.

A “solid carriers” as described by this invention refers to an objectthat has a surface that is so broad that specific areas can bedistinguished upon it. This surface is preferably flat and larger than 5mm², and is preferably between 10 mm² and approx. 100 cm². The carriermaterial is not liquid or gaseous, and preferably dissolves either notat all or incompletely in the sample fluids or reaction preparationsthat are used to immobilize nucleic acids to the surface. Examples ofsuch materials are glass, plastics (e.g. polystyrene, polyamide,polyethylene, polypropylene), gold, etc. The material does notnecessarily have to be completely solid itself, but rather can be madesolid by the attachment of supporting materials.

The external shape of the solid carrier basically depends on the methodused to detect the presence of nucleic acids on this solid carrier. Ithas proven to be appropriate, for instance, to select a basically planarform, e.g. a chip.

Solid carriers that are especially suitable are, therefore, polystyrenechips that are from 1 to 5 mm thick and have a surface area of from 1 to5 cm², for instance. Polyamide membranes that are 4×2.5 cm² in size haveproven to be especially well-suited for use with this invention. Two ormore nucleic acid analogs having different base sequences are bound todifferent sites of the surface of this carrier. These sites or regionspreferably do not overlap with each other. They are preferably separatedfrom each other by regions on the surface to which no nucleic acidanalogs are bound. The sites to which the nucleic acid analogs are boundare referred to as “binding regions” below. The binding regions can havedifferent shapes. These shapes are basically determined by the method ofmanufacturing the solid carrier or by the method used to determine thenucleic acid analogs in the binding regions. The minimum size of thebinding regions is basically determined by the instrument with which theevent—the binding of a nucleic acid to nucleic acid analogs of aregion—is detected. Instruments are already available that can detectbinding to regions that are approx. 1 mm in size. The upper limit of thesize of the binding regions is determined by cost effectiveness andhandling considerations.

The size of the binding regions is also basically determined by themethods used to apply the nucleic acid analogs to the surface. Suchmethods will be described later.

The number of binding regions on the solid carrier depends on theintended use of the solid carrier. In the simplest case, just twobinding regions are needed to detect a certain point mutation. In thiscase, a binding region contains nucleic acid analogs that have a base inthe position at which the point mutation is to be detected. This base iscomplementary to the base in the position of the normal sequence. Theother binding region, on the other hand, contains a nucleic acid analogthat has a base in the corresponding position that is complementary tothe base of the mutated sequence. In another case, two nucleic acids ornucleic acid sequences that are only slightly related to each other canbe detected simultaneously using a solid carrier that has two completelydifferent nucleic acid analogs bound to its surface.

“Nucleic acid analogs” refer to non-naturally occurring molecules thatcan detect nucleic acids by means of base pairings. They thereforecontain a specific base sequence that is completely complementary to thebase sequence of a nucleic acid to be detected. The base sequence istherefore preferably composed of two naturally occurring nucleobases. Aslong as the specificity of the base pairing is not lost, modificationsto the nucleobases are also allowed, however.

Those nucleic acid analogs are considered complementary to a nucleicacid that have a base sequence that forms hydrogen bridges with a basesequence of the nucleic acid per the principle of base pairing when itis bound to the nucleic acid. This sequence is preferably at least 8bases long and, more preferably, between 8 and 25 bases long.

The nucleic acid analogs are further defined by the fact that they arestructurally different from nucleic acids, at least in terms of thebackbone. The “backbone” in nucleic acids or a nucleic acid analogrefers to a structure that is basically composed of identical units thateach contain a base. In naturally occurring nucleic acids, the backboneis a sugar phosphate backbone. This backbone is structurally modified innucleic acid analogs, e.g. in that the sugar or phosphate portion iscompletely or partially replaced with other chemical units such asnon-cyclic components. Basically, identical units can also replace eachother in the backbone.

A few characteristics of nucleic acid analogs are described below tofacilitate the selection of nucleic acid analogs that are suitable foruse with this invention, It is advantageous for nucleic acid analogs tohave a higher affinity to sequence-complementary nucleic acids than anoligonucleotide with an identical base sequence. In addition, thosenucleic acid analogs are preferred that carry fewer charges than acorresponding oligonucleotide of the same length, or that can compensatecharges with the opposite charges. Basically, uncharged nucleic acidanalogs are especially preferred. Especially preferred nucleic acidanalogs are those whose affinity to complementary nucleic acidsbasically does not depend on the salt content of the hybridizationcomplex.

The nucleic acid analogs that are especially suitable are the nucleicacid analogs described in WO 92/20702 and WO 92/20703 (Peptide NucleicAcid, PNA, e.g. Nature 365, 566–568 (1993) and Nucl. Acids Res. 21,5332–5 (1993)). These patent applications are referred to for thedescription of the structure of the nucleic acid analogs. Preferrednucleic acid analogs are compounds that have a polyamide backbone thatcontains a number of bases bound along the backbone, with each basebound to a nitrogen atom of the backbone. Nucleic acid analogs shouldalso include compounds, however, like those described in EP-A-0 672 677.Additional nucleic acid analogs are described in Recueil 91, 1069–1080(1971), Methods in Molecular Biology 29, 355–389 (1993), Tetrahedron 31,73–75 (1975), J. Org. Chem. 52, 4202–4206 (1987), Nucl. Acids Res. 17,6129–6141 (1989), Unusual Properties of New Polymers (Springer Verlag1983), 1–16, Specialty Polymers (Springer Verlag 1981), 1–51, WO92/20823, WO 94/06815, WO 86/05518 and WO 86/05519. Additional nucleicacid analogs are described in Proc. Natl. Acad. Sci. USA 91, 7864–7868(1994), Proc. Nat. Acad. Sci. USA 92, 6097–6101 (1995) and J. Am. Chem.Soc. 117, 6140–6141 (1995). The nucleic acid analogs described are from8 to 30 bases long, while a length of from 10 to 25 bases is especiallyadvantageous. The nucleic acid analogs named are bound directly orindirectly to the surface of the solid carrier. The type of bindingbasically depends on which reactive groups are available for binding onthe solid carrier, and which reactive groups are available for bindingto the nucleic acid analog without restricting the ability of thenucleic acid analogs to bind to a complementary nucleic acid. The typeof binding also depends on whether the intent is to simultaneously bindthe nucleic acid analogs to different sites, or to build upon them. Itcan also be appropriate to cover the surface of the solid carrier with alayer of a material that has a greater ability to bind, or to activatethe surface by means of a chemical reaction. Reactive groups on thesurface of a solid carrier are usually selected from the group —OH, —NH₂and SH. Reactive groups of nucleic acid analogs are preferably selectedfrom the group —OH, NH₂, —SR, —COOH, —SO₃H and —PO₃H₂.

In an especially preferred embodiment, the reactive groups of thesurface and the nucleic acid analog are covalently bound to each other,especially by means of a linker that is more than 15 atoms and less than200 atoms long. A “linker” refers to a portion of a molecule thatbasically has the function of removing the nucleic acid analogs that aresterically available on the surface of the solid carrier. A linker isusually selected that has hydrogen atoms (e.g. in alkylene units) andnumerous heteroatoms (e.g. —O— or —NH— or —NR—) that facilitatesolvation. The linker preferably contains one or more ethylene oxy unitsand/or peptide groups. In an especially preferred embodiment, the linkercontains one or more units as described in DE-A 3924705. Especiallypreferred are the units described as an example which is referred to asAdo (8-amino-3,6-dioxa-octanic acid), below. A slight dependence of thebinding of nucleic acids to the PNA surface can be reduced by usinglonger linkers between PNA and the solid carrier.

The nucleic acid analog that is bound to a site can also be a mixture oftwo or more analogs having different but known sequences. This canreduce the number of sites required for a multiple determination.

The surface of the carrier is preferably not charged, and is preferablyhydrophilic. The invention demonstrated that the use of basicallyuncharged surfaces is an advantage when detecting nucleic acids.

A solid carrier loaded with nucleic acid analogs at different sites asprovided by this invention can be manufactured in different ways. In oneembodiment, suitable quantities of solutions that each contain differentnucleic acid analogs are applied to different sites on the surface ofthe solid carrier, e.g. via pipette. The liquid samples should not mixwith each other on the surface of the solid carrier. This can beaccomplished, for instance, by locating the application sites far apartfrom each other or by using a hydrophobic barrier to stop the expansionof the liquid between the various sites. Either the nucleic acid analogsor the surface of the solid carrier is preferably activated for thereaction. This activation can be achieved, for instance, in that one ofthe groups described above is activated by the creation of a reactivespecies. In the case of a carboxyl group, this would be an activatedester (e.g. a N-hydroxy succinimide ester) that quickly enters into anester bond with a hydroxyl group without further activation. Suitablyactivated polyamide membranes carry triazine groups, for instance, thatcan react with amino groups of nucleic acid analogs with the formationof a covalent bond. The activation can also take place by means ofbifunctional reagents, squaric acid derivatives of WO 95/15983 orglutaraldehyde (GB 2197720).

The binding of the analogs can also be realized by coating the carriersurface with nucleotide sequences that are complementary to a part ofthe sequence of the nucleotide analogs. The binding of the differentnucleic acid analogs to the binding regions can take placesimultaneously or sequentially.

After a sufficient amount of time has passed for the binding to takeplace, it is advantageous to wash away any nucleic acid analogs thathave not bound or are bound insufficiently, along with any bindingreagents that were used. This is performed preferably under conditionsin which non-bound nucleic acid analogs cannot bind with nucleic acidanalogs that are bound to other regions.

Basically, it is also possible to build upon the nucleic acid analogs onthe different sites of the surface by means of monomeric units. Thetechnology described in WO 92/10092 or WO 90/15070 can be used for thispurpose. Appropriate monomers are described in WO 92/20702, forinstance.

Another subject matter of this invention is a method for thesequence-specific detection of a nucleic acid using the solid carrierprovided by this invention.

Detectable nucleic acids are natural or artificial nucleic acids.“Nucleic acids” therefore also refer to nucleic acid analogs. Thenucleic acid to be detected, however, is the RNA or DNA in particularthat is characteristic for an organism containing nucleic acids, e.g. avirus, a bacterium, a multicellular organism, a plasmid or a geneticcondition such as a predisposition or a disposition for a certaindisease or a spontaneous genetic mutation. The RNA and DNA in this caseis basically of genomic origin or an origin derived therefrom. Animportant class of nucleic acids in the context of this invention arethe results of a nucleic acid amplification. These results are alsoreferred to as “amplificates” or “amplicons” below. The nucleic acidscan be present in either their raw form or in a purified or processedform. A purification can also take place by separating the nucleic acidsfrom cell components in a preparatory step, e.g. an affinity separationstep. The nucleic acids can also be enzymatically extended, specificallyamplified or transcribed.

For the sequence-specific detection of nucleic acids, the carrierprovided by this invention has a nucleic acid analog bound to a sitethat has a base sequence that is complementary to a base sequence of thenucleic acid to be detected. This base sequence is selectedintentionally so that it can specifically reveal the presence of thenucleic acid. In the normal case this means that the mixture contains asfew as possible—and preferably no—additional nucleic acids having thesame total sequence. It must be mentioned, however, that the carrierprovided by this invention can also be used to specifically detectgroups of nucleic acids. It can be a task of the method, for instance,to detect any member of a certain taxonomic group, e.g. a family ofbacteria, by means of its nucleic acids. In this case, the base sequenceof the nucleic acid analog can be intentionally selected so that it liesin a conserved region but only occurs in members of this taxonomicgroup.

An additional nucleic acid analog that has a base sequence that is notcomplementary to the same base sequence is preferably bound to adifferent site of the surface. It can be a nucleic acid analog, the basesequence of which can be shorter or longer, or which can differ from thefirst nucleic acid analog by one or more bases. The difference in thebase sequence depends on the task to be solved. The differences caninclude point mutations, or smaller deletions and insertions, forinstance. In many genetic diseases, such as cystic fibrosis, thesequences of the nucleic acid analogs differ in terms of individualpositions (point mutations) and numerous positions (deletions, forexample at Δ 508).

The mutation to be detected is preferably positioned close to the middleof the base sequence of the analog.

The sequences of the analog can also be intentionally selected so thattheir hybridization positions differ by one base each, even though thelengths are identical (overlap). The sequences can also be intentionallyselected so that the hybridization regions are adjacent to each other onthe nucleic acid to be detected.

Numerous mutations can also be detected in the same or different nucleicacids by selecting nucleic acid analogs with sequences that arecomplementary to the sequence on these nucleic acids.

In an especially easy case in which the purpose is to determine which oftwo alleles is present in a complex, two nucleic acid analogs are boundto different sites of the surface of the solid carrier, the basesequences of which differ in exactly that position where the allelesalso differ. A nucleic acid analog is therefore selected that iscomplementary to a certain sequence of one allele, while the othernucleic acid analog is complementary to the sequence of the otherallele. The length and hybridization sites of the nucleic acid analogsare identical.

All alleles are usually detected for cystic fibrosis, for instance. Thewild-type contains two healthy alleles. Heterozygotes contain onemutated and one wild-type sequence, and homozygotic mutants contain twomutant nucleic acids. In this case, the intent is not just to determineif mutants are present, but to determine if it is a heterozygotic orhomozygotic case. In accordance with this invention it is possible tosimultaneously quantitatively detect both alleles and therebydifferentiate between the three cases described.

In many cases, especially in oncology and in the determination ofinfectious parameters, mutated cells/particles are usually located inthe background of non-mutated/normal cells. In these cases, selectivedetection can not be performed reliably or at all using methods providedby the state of the art. The analysis of ras mutations from DNA fromstool samples, for instance, requires that a mutated sequence bereliably detected in the presence of approx. 100 normal sequences(Science 256, 102–105 (1992)). In the field of infectious diseases, itwould be desirable to determine different HIV populations in oneinfected patient. The quantity of many mutants of these HIV populationsis less than 2% compared with all HIV sequences, however. The methodprovided by this invention therefore makes it especially quite possibleto investigate mixtures of nucleic acids that are very similar to eachother, even if one of the nucleic acids is present in a much greaterquantity than the nucleic acid to be determined.

The lengths of the bound nucleic acid analogs are preferably identical.An appropriate length has proven to be between 10 and 100, andpreferably between 10 and 50 bases. Especially good results are obtainedwith nucleic acid analogs that are between 10 and 25 bases long.

To perform the method provided by this invention, the sample containingnucleic acid is brought in contact with the sites on the surface of thecarrier that have bound the nucleic acid analogs. This can be performed,for instance, by bringing the solid carrier into the sample fluid orpouring the sample fluid onto the solid carrier in one or more portions.The nucleic acids in the sample fluid can be denatured (single-strand)before they are brought in contact with the carrier. A major advantageof the invention, however, is the fact that a preliminary denaturationstep can be eliminated, e.g. by using PNAs. The PNAs force a strand outof the double-stranded nucleic acid to be determined. The only importantrequirement is that the sample be brought in contact with the solidcarrier under conditions in which the nucleic acid to be detected bindsspecifically to the appropriate site on the surface by means of thenucleic acid analog which is complementary to one sequence of thenucleic acid to be determined. These conditions can be different fordifferent types of nucleic acid analogs, of course, but they are easilydetermined for given nucleic acid analogs by performing tests. In thenormal case, these conditions are based on the conditions that are knownfor carriers loaded with oligonucleotides. If nucleic acid analogs areused as described in WO 92/20702, however, conditions can be selectedthat are much different from the hybridization conditions for thecorresponding oligonucleotides. It has proven to be appropriate,however, to use much less salt than when the correspondingoligonucleotides are used. For instance, the presence of less than 100mM and, more preferably, less than 50 mM, and most preferably, less than10 mM salt is recommended. Under these conditions, it would not bepossible to sufficiently differentiate between nucleic acids havingsimilar sequences using oligonucleotides having the same sequence.

The sample is kept in contact with the surface as long as necessary toachieve a sufficient binding of the nucleic acids to the appropriatesite on the surface. This period is usually a few minutes.

In the next step, it is determined whether the nucleic acid has bound tothe surface and, if so, to which site. This is considered an indicationof the presence of a nucleic acid that contains a base sequence that iscomplementary to the nucleic acid analog bound to this site. The bindingcan be determined using various methods.

Instruments are already available with which changes on specific sitesof surfaces can be determined directly. For methods that use these typesof instruments it is not even necessary to remove the sample containingthe nucleic acid from the surface after it has been applied. Normally,however, it is preferable to remove the fluid from the surface and use awash solution to remove any remaining reagent that is still adhered tothe surface. This step provides the advantage of also washing awaysample components that can interfere with the determination of thebinding.

In a preferred embodiment, the binding of the nucleic acid to bedetected with the nucleic acid analog is determined by means of a labelthat is inserted in the nucleic acid to be determined in a step that isperformed before the sample is brought in contact with the surface. Thelabel can be a detectable group such as a fluorescent group, forinstance. This determination can be performed optically using amicroscope or in a measuring cell provided for this purpose. While thesite at which the binding took place is an indication of the presence ofa nucleic acid having a certain sequence, the quantity of label at apredetermined site can be used as an indication of the quantity of thenucleic acid to be determined.

In an especially preferred embodiment, the nucleic acids to be detectedare the products of a nucleic acid amplification method such as thepolymerase chain reaction as described in EP-B-0 202 362, or NASBA asdescribed in EP-A-0 329 822. It is important that the nucleic acidsequence to bind with the nucleic acid analog be amplified by theamplification method. The better the amplification method maintains theoriginal sequence—that is, the fewer errors that are incorporated intothe sequence during amplification—the more suitable the amplificationmethod. The polymerase chain reaction has proven to be especiallysuitable. The amplification primers are selected specifically so thatthe nucleic acid sequence to be detected lies in the region between thehybridization sites.

It has also proven advantageous to insert the label required for thedetection reaction into the amplificate during amplification. This canbe performed, for instance, by using labelled primers or labelledmononucleoside triphosphates.

The binding that took place can be detected directly without inserting alabel, for instance, by using an intercalating agent. These agents havethe characteristic of depositing selectively on double-strandedcompounds that contain bases, including the complex of the nucleic acidanalog and the nucleic acid bound in sequence-specific fashion. Thepresence of the complexes can be detected using specific characteristicsof intercalating agents, e.g. fluorescence. Ethidium bromide is anespecially suitable agent.

Another method for detecting hybrids without inserting a label is basedon surface plasmon resonance, as described in EP-A-0 618 441, forinstance.

According to another possible method for determining the binding, thesurface is brought in contact with a solution of an antibody labelledfor detection. This antibody is directed against the complex consistingof the nucleic acid analog and the nucleic acid to be determined.Antibodies of this nature are described in WO 95/17430, for instance.

The detection of the hybrids depends on the type of labelling used. Thehybrids can be detected with a scanner, a CCD camera or a microscope,for instance.

This invention provides numerous advantages. In particular, it allowsdetecting sequence differences in nucleic acid regions located withinsecondary structures. With this invention it is also possible toincrease the sensitivity of detection, because it can use a greaterabsolute quantity of bound nucleic acid to be determined thantraditional methods. It is also possible, in particular, to increase thesignal-to-noise ratio compared with methods that use oligonucleotides.In the first attempt, a signal-to-noise ratio of less than 1:1000 wasobtained.

The invention can be used in at least two fields. In the first case, thesolid carrier is used to detect known mutations and polymorphisms. Inthis case, the number of mutations and polymorphisms to be determined isan indicator of the number of different nucleic acid analogs or sitesrequired on the surface. The sequence of the nucleic acid analogs isspecially coordinated with the sequence of the nucleic acids around themutations and polymorphisms. Preferably, the sequences are selected insuch a way that the base by which nucleic acid analogs having similarsequences differ is located in or near the middle of the sequence.

The carriers provided by this invention can be used in the followingfields:

Infectious diseases, the simultaneous determination of differentanalytes/parameters, and in investigations of the condition of a gene ina bacterium or virus, e.g. for multi-drug resistance studies.

Oncology (detection of mutations in tumor suppressor genes andoncogenes, and in the determination of the relationship between mutatedand normal cells).

Investigation of inherited diseases (cystic fibrosis, sickle cellanemia, etc.).

Tissue and bone marrow typing (MHC complex) (see Clin. Chem. 41/4, 553–5(1995)).

In a second potential application, a sequence of short nucleic acidfragments can be determined using the method called “sequencing byhybridization”. In this method, the same number of different nucleicacid analogs are immobilized as there are permutations of the selectedlength of the sequence. To achieve a sufficient level of sensitivity,4^(N) sites are required, with N equal to the number of bases in eachnucleic acid analog. Preferably, N is between 5 and 12. Correspondinglyfewer sites are required to sequence very short DNA fragments. Themethod for sequencing unknown nucleic acids using the “sequencing byhybridization” method is described in WO 92/10588.

An advantage of this invention is the fact that the specificity of thehybridization is largely independent of the conditions in the sample.This facilitates simultaneous binding of nucleic acids to differentregions on the surface.

Surprisingly, it was shown that nucleic acid analogs such as PNA have anexcellent ability to discriminate between sequences on the surface. Thisdiscrimination was better than was to be expected from the meltingtemperatures of analogous, dissolved compounds. Surprisingly, thecarriers provided by this invention are even suitable for use innumerous, consecutive determinations of nucleic acids. After adetermination is performed, the carrier that is in contact with a fluidundergoes heat treatment. A temperature is selected at which the bondbetween the nucleic acid analog and the nucleic acid is dissolved. Thecarrier is then available to perform another determination. With themethod provided by this invention, it is possible for the first time todetermine relative quantities of very similar nucleic acids located nextto each other in a sample in a concentration range of at least two logs.It has been possible to quantitatively determine mutants usingsequencing methods, for instance. The maximum level of discriminationavailable with this method was 1:10, however.

With the method provided by this invention it is also possible to binddouble-stranded DNA to the immobilized nucleic acid analogs of the solidcarrier without a denaturation step. Comparison studies have shown thatthis is not possible with immobilized DNA. It has also been shown thatmismatches that are not located in the center of the hybrid can also bedistinguished with a high degree of selectivity.

The sequences of the PNA molecules are shown in FIG. 1 a. They are usedas examples to explain the method. The PNAs were prepared as describedin WO 92/20702.

FIG. 1 b shows the sequences of the DNA molecules that are homologous tothe PNA sequences from FIG. 1 a and that were used for the DNA/ODNhybridization experiments.

FIG. 1 c shows the sequences of the complementary oligonucleotides (ODN)used that were labelled with digoxigenin on the 5′-phosphate end usingthe 5′-DIG End Labelling Kit (Boehringer Mannheim) and that werephosphorylated with polynucleotide kinase and ³²P-g-ATP 5′.

FIG. 1 d shows the feasible combinations of oligonucleotide (ODN) andPNA for forming hybrids. Identical combinations apply for hybridizationsbetween DNA probes (DNA, see FIG. 1 b) and oligonucleotides (ODN, seeFIG. 1 c).

FIG. 2 shows the hybridization results from Example 4 to illustrate theselectivity of the method. The conditions were: 200 nl spot volume (100;10; 1; 0.1 mM PNA, one concentration per cleavage), incubation at 45° C.

FIG. 3 shows the hybridization results from Example 6. They verify thatPCR amplicons are detected by immobilized PNA probes. The conditionswere: 200 nl spot volume (100; 10; 1; 0.1 mM PNA, one concentration percleavage), incubation at 45° C. The labels in FIG. 3 mean:

-   I Control experiment, ODN 1 a (1 pMol, 1 nM), line I (PNA 1), line 2    (PNA 2) line 3 (PNA 3)-   II ss Amplificate (118 bp, one-fold DIG-labelled) 5 min heat    denaturation at 94° C. (50 ml PCR preparation diluted in 1 ml    hybridization buffer) Line 1 (PNA 1), line 2 (PNA 2), line 3 (PNA 3)-   III ds Amplificate (118 bp, one-fold DIG-labelled) (50 ml PCR    preparation diluted directly in 1 ml hybridization buffer) Line 1    (PNA 1), line 2 (PNA 2), line 3 (PNA 3)

FIG. 4 shows the results of the qualitative and quantitative analysis ofanalyte mixtures by means of PNA arrays. The conditions were: 200 nlspot volume (100 mM PNA, one PNA per line, one analyte mixture percleavage).

FIG. 5 shows the effect of linker length on the hybridization. Theconditions were: 1 ml spot volume (100; 40; 20; 10; 5; 1 mM PNA, oneconcentration per cleavage, (Ado)₃-PNA in row 1, (Ado)₆-PNA in row 2,(Ado)₉-PNA in row 3). The labels in FIG. 5 mean:

-   A. Prehybridization/hybridization in 5 mM sodium phosphate, 0.1%    SDS, pH 7.0-   B. Prehybridization/hybridization in 10 mM sodium phosphate, 0.1%    SDS, pH 7.0-   C. Prehybridization/hybridization in 25 mM sodium phosphate, 0.1%    SDS, pH 7.0

FIGS. 6 a–6 c demonstrate that PNA-derivatized membranes can be usedmany times after regeneration. The conditions were: 1 ml spot volume(100; 40; 20; 10; 5; 1 mM PNA, one concentration per cleavage).

The labels in FIG. 6 mean:

-   -   6 a: Signal intensities after the first hybridization    -   6 b: Signal intensities after the regeneration procedure    -   Membrane 1: No regeneration (controls)    -   Membrane 2: Regeneration with 0.1 M sodium hydroxide solution,        RT 1 h, 2×10 min bidistilled water RT    -   Membrane 3: Regeneration with 1 M sodium hydroxide solution, RT,        1 h, 2×10 min bidistilled water RT    -   Membrane 4: Regeneration with distilled water, 70° C. 1 h, 2×10        min bidistilled water RT    -   Membrane 5: Regeneration with 0.1 M sodium hydroxide solution,        70° C. 1 h, 2×10 min bidistilled water RT    -   6 c: Signal intensities after rehybridization

This invention is explained in further detail using the followingexamples:

EXAMPLES General

The nucleic acid analogs used were manufactured as described in WO92/20702. Unless indicated otherwise, chemicals and reagents wereproducts of Boehringer Mannheim GmbH.

Example 1 Covalent Derivatization of Nylon Membranes

200 nl of a solution that contains PNA in the desired concentration in0.5 M sodium carbonate pH 9.0 are applied to an Immunodyne ABC membrane(Pall) with a pipette. After the spots are dry, the membrane is washedwith 0.1 M sodium hydroxide solution to deactivate any reactivefunctional surface groups that may still be present. The membrane iswashed a second time with water and then dried.

Example 2 Detection of a Hybridization Event Using Luminescence

The membrane is derivatized as described in Example 1 using 100 μM, 10mM, 1 μM and 0.1 mM PNA solutions. It is then prehybridized in a 50 mlhybridization vessel with 10 ml hybridization buffer (10 mM sodiumphosphate, pH 7.2, 0.1% SDS (sodium dodecylsulfate)) in a hybridizationoven at 45° C. After 30 minutes, 10 ml of a solution that contains theDIG-labelled oligonucleotide in a 1 mM concentration is added and thecomplex is hybridized for another 60 minutes. It is then washed for 2×10minutes with 25 ml wash buffer each time (5 mM sodium phosphate pH 7.2,0.05% SDS) at 45° C. The detection reaction is performed according tothe protocol for digoxigenin detection (DIG Detection Kit, BoehringerMannheim GmbHI BRD). The anti-DIG-AP conjugate is used in a 1:10000dilution. CDP-Star™ is used in a 1:10000 dilution as the substrate forthe alkaline phosphatase.

Example 3 Detection of a Hybridization Event Using Fluorescence

The membrane is derivatized as described in Example 1 using 100 μM, 10μM and 1 μM PNA solution. The membrane is prehybridized in a 50 mlscrew-top container with 10 ml hybridization buffer (see Example 2) inthe oven at 45° C. After 30 minutes, 10 ml of a solution that contains afluorescent-labelled oligonucleotide in a concentration of 1 μM isadded, and the preparation is hybridized for another 60 minutes. Themembrane is then washed for 2×10 minutes with 25 ml wash buffer eachtime (see Example 2) at 45° C. The membrane is dried, then the intensityof the fluorescence is measured.

Example 4 Selectivity of the Method

Three membrane strips are derivatized with three (Ado)₆-PNA moleculeseach that differ according to one or two positions of their basesequence (see FIG. 1 a, SEQ. ID. NOS. 1, 2, 3), using PNA solutions in aconcentration range of between 100 mM and 0.1 mM as described inExample 1. The membrane strips are prehybridized with 10 mlhybridization buffer for 30 minutes in 50 ml screw-top containers. Inthe next step, one of the three DIG-labelled oligonucleotides (FIG. 1 b,SEQ. ID. NOS. 4, 5, 6) is added. After hybridizing for 60 minutes, themembranes are washed for 2×10 minutes with 25 ml wash buffer each time.The hybridization events are detected as described in Example 2.

All possible double-stranded hybrids between the PNA molecules involvedand the oligonucleotides are shown in FIG. 1 d. FIG. 2 illustrates that,in almost every case, the only oligonucleotide detected is the one thatis exactly complementary to the immobilized nucleic acid analog (PNA 1,PNA 2, PNA 3). The signal-to-noise ratios (S/N) can also be estimatedfrom the figure. They were evaluated quantitatively, and the results arepresented in Table 1.

TABLE 1 Hybrid (PNA/ODN) S/N Signal (Hybrid)/Signal (Match) 1/1 655.2100.0% 2/1 20.7  3.2% 3/1 10.3  1.6% 1/2 23.1  2.6% 2/2 871.8 100.0% 3/26.4  0.7% 1/3 109.4  22.7% 2/3 12.3  2.5% 3/3 481.1 100.0%

Example 5 Quantification

Membrane strips are derivatized with three (Ado)₆-PNA molecules withdifferent base sequences (FIG. 1 a, SEQ. ID. NOS. 1, 2, 3) in aconcentration of 100 mM as described in Example 1. They are thenprehybridized in 20 ml hybridization vessels with 10 ml hybridizationbuffer (see Example 2) at 45° C. The buffer is replaced after 30minutes. In experiments 1 through 7, the buffer to be added differsaccording to the analyte concentrations of the DIG-labelledcomponents—oligonucleotide 1, 2 and 3, SEQ. ID. NOS. 4, 5, 6. The stripsare hybridized for 60 minutes at 45° C. and then washed for 2×10 minuteswith 10 ml wash buffer. The detection is performed using the proceduredescribed in Example 2. The luminescence signal is recorded with aluminescence imager and then evaluated (FIG. 4).

The signal intensities found can be used to reach a qualitative orsemi-quantitative finding regarding the composition of the analytecomplex. Absolutely quantitative findings can be reached after thesignal intensities are calibrated.

Example 6 Detection of PCR Amplicons

A. Obtaining a Suitable Analyte (Amplificate)

A double-stranded DNA fragment is ligated in a pUC19 plasmid, thesequence of which is complementary to the PNA probe PNA 1. The plasmidis transformed in E. coli, cloned, and then sequenced. For thesubsequent hybridization experiments, a section of the plasmid sequenceis amplified and DIG-labelled during the amplification reaction. Theamplification is performed in a total volume of 50 μl. The amplificationcomplex consists of 1 μl plasmid (1 ng/μl) 1 μl primer F1 (10 μM), 1 μlDIG primer R1 (10 μM), 5 μl 10×PCR buffer (100 mM Tris/HCl, 15 mM MgCl₂,500 mM KCl, pH 8.3), 2 μl dNTP solution (10 mM dATP, 10 mM dCTP, 10 mMdGTP, 10 mM dTTP in distilled water, pH 7.0), 0.5 μl Taq polymerase (5units/μl) and 38.5 ml water.

Primer F1: 5′-GTA AAA CGA CGG CCA GT-3′ (SEQ.ID.NO.12) Primer R1:5′-DIG-AAC AGC TAT GAC CAT GA-3′ (SEQ.ID.NO.13)

Each reaction mixture is warmed to 96° C. for 3 minutes. In the nextstep, 30 rounds of a 3-level PCR cycle are performed (45 sec. 96° C., 30sec, 48° C., 1 min 72° C.). In the last cycle, the elongation step isincreased by 5 minutes at 72° C.

B. Hybridization Reaction

The membranes are derivatized with three (Ado)₆-PNA sequences each thatdiffer according to one or two positions in their base sequence (seeFIG. 1 a, SEQ. ID. NOS. 1, 2, 3) using PNA solutions in a concentrationrange between 100 μM and 0.1 μM as described in Example 1. The membraneis pretreated in a 20 ml hybridization vessel with 5 ml hybridizationbuffer at 45° C. The buffer is replaced after 30 minutes and the analytesolution is added. To make the analyte solution, the amplificationcomplex is diluted directly (ds amplicon) and, after 5 minutes of heatdenaturation (ss amplicon), in 1 ml hybridization buffer. Afterhybridization for 1 h, 2 h 30 min and 4 h at 45° C., the membranes arewashed for 2×10 minutes with 5 ml wash buffer each time. Hybridizationevents are detected as described in Example 2 (FIG. 3).

Nine fields are shown in FIG. 3. The difference between each row is theincubation period (4 h, 2½ h, and 1 h). The difference between eachcolumn is the type of nucleic acid to be detected. Three overlappingrows of spots are applied to each of the 3 fields of column I. Thedifference between the rows is the sequence of the PNAs, while thedifference between the columns of each field is the concentration. Thespecificity and the ability to be quantified are indicated in column Ifor the case in which oligonucleotides are used as the detecting nucleicacid.

The figure illustrates the influence of incubation time. It is clearthat an excellent sequence discrimination for ODN 1 a and theamplificates is obtained after hybridization for just one hour. Thedifference between columns II and III in FIG. 3 is that an amplificatethat was previously made single-stranded is used in one case as thenucleic acid to be detected. In column III, an amplificate that was notpreviously made single-stranded is used as the nucleic acid to bedetected. The signals indicate clearly that it is not necessary todenature double-stranded nucleic acids before applying them to the solidcarrier. This decreases the number of working steps (heating step,single strand separation, wash step) and, therefore, reduces the dangerof contamination. PNA probes in combination with low-salt conditionstherefore offer clear advantages over DNA probes.

Example 7 Comparison of PNA/DNA Hybridization

Membrane strips are derivatized with three (Ado)₆-PNA sequences each(SEQ. ID. NOS. 1, 2, 3) and three DNA molecules each (SEQ. ID. NOS. 8,10, 11) that differ according to one or two positions in their basesequence (see FIGS. 1 a and 1 b) using 50 mM solutions as described inExample 1. Unlike Example 1, the spot volume is 400 nl instead of 200nl. The membrane strips are prehybridized in 20 ml hybridization vesselswith either 5 ml low-salt buffer (see Example 2) or high-salt buffer(6×SSC; 0.9 M NaCl, 90 mM sodium citrate, 0.1% SDS, pH 7.0) at either37° C. or 45° C. for 30 minutes. In the next step, one of the threeDIG-labelled oligonucleotides (FIG. 1 c, SEQ. ID. NOS. 4, 5, 6) isadded. After a hybridization step of 60 minutes, the strips are washedfor 2×10 minutes with 5 ml wash buffer each time at 37° C. or 45° C. Thewash buffer from Example 2 is used for the low-salt experiments. For thehigh-salt experiments, a 1×SSC buffer with 0.02% SDS, pH 7.0 is used.The hybridization results are detected as described in Example 2. Theevaluation is performed quantitatively and is illustrated in Table 2.

Both the DNA and PNA probes are able to completely discriminate betweencomplementary, single-stranded target sequences of single and doublemismatched sequences. PNA probes demonstrate clear advantages over theDNA probes for certain types of mismatches, especially when they are notlocated in the middle of the sequence, but rather shifted to the end.This becomes especially clear in the example of a decentral G/T mismatch(probe 1/ODN 3), which is tolerated by the DNA probe much more stronglythan by the PNA probe having the identical sequence.

TABLE 2 PNA PNA PNA or DNA 1 or DNA 2 or DNA 3 ODN 1 PNA 45° C., lowsalt 100.0%  1.2%  1.5%  DNA 37° C., high 100.0%  1.2%  6.4%  salt ODN 2PNA 45° C., low salt 1.1%  100.0%  2.9%  DNA 37° C., high <2%* 100.0% <2%* salt ODN 3 PNA 45° C., low salt 26.0%  1.5%  100.0%  DNA 37° C.,high 66.5%  <2%* 100.0%  salt *A more exact value cannot be determinedbecause the spot intensity is lower than the standard deviation of thebackground signal.

Example 8 Influence of the Length of the Linker Between the Membrane andPNA Probes

Membrane strips are derivatized with PNA molecules (see FIG. 1 a, SEQ.ID. NOS. 7, 1, 9) that differ according to the length of the linker(Ado₃, Ado₆ or Ado₉) using PNA solutions in the concentration rangebetween 100 μM and 1 μM as described in Example 1. Unlike Example 1, thespot volume is 1 μl instead of 200 nl. The membrane strips areprehybridized in hybridization containers with 10 ml hybridizationbuffer (5, 10 or 25 mM sodium phosphate, 0.1% SDS, pH 7.0) for 30minutes at 35° C. In the next step, 10 pMol ³²P-labelled oligonucleotide(see FIG. 1 c: ODN 1 b, SEQ. ID. NO. 4) is added and the preparation ishybridized for 60 minutes at 50° C. The membranes are washed for 2×10minutes with 50 ml wash buffer (5 mM sodium phosphate, 0.1% SDS, pH 7.0)at 50° C. The hybridization events are detected using autoradiography(FIG. 5). The figure shows that a longer linker greatly improves thehybridization.

Example 9 Reuse of PNA Membranes

A membrane is derivatized with (Ado)₆-PNA molecules (see FIG. 1 a: PNA 1b, SEQ. ID. NO. 1) using PNA solutions in a concentration range between100 μM and 1 μM as described in Example 1. The PNA is applied in fiveidentical concentration sequences. The spot volume is 1 μl, as inExample 8. The membrane is prehybridized in a hybridization vessel with10 ml hybridization buffer (10 mM sodium phosphate, 0.1% SDS, pH 7.0)for 30 minutes at 35° C. In the next step, 10 pMol ³²P-labelledoligonucleotide (see FIG. 1 c: ODN 1 b, SEQ. ID. NO. 4) is added and thepreparation is hybridized for 60 minutes at 50° C. The membrane iswashed for 2×10 minutes with 50 ml wash buffer (5 mM sodium phosphate,0.1% SDS, pH 7.0) at 50° C. The hybridization events are detected usingautoradiography (FIG. 6 a). After the autoradiography is performed, themembrane is cut into five identical strips. These membrane strips areeach treated differently in the rest of the experiment. Membrane 1 isnot incubated and serves as the control membrane. Membrane 2 isincubated for 60 minutes at room temperature with 50 ml 0.1 M sodiumhydroxide solution. Membrane 3 is incubated for 60 minutes as well, with50 ml 1 M sodium hydroxide solution. Membrane 4 is incubated for 60minutes at 70° C. with 50 ml distilled water. Membrane 5 is incubatedfor 60 minutes at 70° C. with 50 ml 0.1 N sodium hydroxide solution. Allmembranes are then washed with distilled water for 2×10 minutes. Afterthis procedure is completed, autoradiography is performed once more(FIG. 6 b). These membrane strips are then used a second time in ahybridization reaction as described, and the hybridization events aredetected using autoradiography (FIG. 6 c).

As shown in FIG. 6 b, the different treatment methods yield verydifferent results. Treatment with bidistilled water at 70° C. (membrane4) causes the membrane to regenerate almost completely. An unexpecteddiscovery was the fact that the success of the regeneration is poorer ifconditions are used that are common for the denaturation of nucleicacids. As such, the incubation of membrane 3 with 1 M sodium hydroxidesolution at room temperature yields virtually no regeneration effect.Decreasing the concentration of sodium hydroxide solution from 1 M to0.1 M increases the degree of regeneration at room temperature (membrane2) and at 70° C. (membrane 5). None of these conditions, however,results in an even slightly good degree of regeneration, as is the casewith bidistilled water (membrane 4). The example shows that theseconditions are important parameters for the efficient denaturation ofmembrane-bound PNA/DNA double-strands.

Regardless of the regeneration method, all membranes can be reused forhybridization (FIG. 6 c), without considerably worsening thesignal-to-noise ratio. Up to 6 rehybridizations could be performedwithout a noticeable effect on the PNA membranes' ability to regenerateor rehybridize.

1. A solid carrier comprising at least two single stranded peptidenucleic acid probes of different base sequence covalently bound to thesolid carrier at separate and distinct regions; wherein the probes havethe general formula (I):

wherein: n is at least 2, each of L¹–L^(n) is independently selectedfrom the group consisting of hydrogen, hydroxy, (C₁–C₄) alkanoyl,naturally occurring nucleobases, non-naturally occurring nucleobases,aromatic moieties, DNA intercalators, nucleobase-binding groups andreporter ligands, at least one of L¹–L^(n) being a naturally occurringnucleobase, a non-naturally occurring nucleobase, a DNA intercalator, ora nucleobase-binding group; each of A¹ through A^(n) is a single bond, amethylene group or a group of formula (IIa) or (IIb):

wherein in (IIa) or (IIb): X is O, S, Se, NR³, CH₂ or C(CH₃)₂; Y is asingle bond, O, S or NR⁴; each of p and q is an integer from 1 to 5, thesum p+q being not more than 10; each of r and ss is zero or an integerfrom 1 to 5, the sum r+ss being not more than 10; each R¹ and R² isindependently selected from the group consisting of hydrogen, (C₁–C₄)alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted,hydroxy, alkoxy, alkylthio, amino and halogen; and each R³ and R⁴ isindependently selected from the group consisting of hydrogen,(C₁–C₄)alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C₁–C₄)alkyl,hydroxy, alkoxy, alkylthio and amino; and wherein in formula (1) each ofB¹–B^(n) is N or R³N⁺, where R³ is as defined above; each of C¹–C^(n) isCR⁶R⁷, CHR⁶CHR⁷ or CR⁶R⁷CH₂, wherein R⁶ is hydrogen and R⁷ is selectedfrom the group consisting of the side chains of naturally occurringalpha amino acids, or R⁶ and R⁷ are independently selected from thegroup consisting of hydrogen, (C₂–C₆) alkyl, aryl, aralkyl, heteroaryl,hydroxy, (C₁–C₆) alkoxy, (C₁–C₆) alkylthio, NR³R⁴ and SR⁵, where R³ andR⁴ are as defined above, and R⁵ is hydrogen, (C₁–C₆)alkyl, hydroxy-,alkoxy-, or alkylthio- substituted (C₁–C₆)alkyl, or R⁶ and R⁷ takentogether complete an alicyclic or heterocyclic system; each of D¹–D^(n)is CR⁶R⁷, CH₂CR⁶R⁷ or CHR⁶CHR⁷, where R⁶ and R⁷ are as defined above;each of G¹–G^(n-1) is

in any orientation, wherein R³ is as defined above; Q is —CO₂H,—CONR′R″, —SO₃H or SO₂NR′R″ or an activated derivative of —CO₂H, —SO₃H;and I is —NHR′″R″″ or —NR′″C(O)R″″, where R′, R″, R′″ and R″″ areindependently selected from the group consisting of hydrogen, alkyl,amino protecting groups, reporter ligands, intercalators, chelators,peptides, proteins, carbohydrates, lipids, steroids, oligonucleotidesand soluble and non-soluble polymers, wherein C and D can optionally beCHR⁶(CH₂)_(sss)CHR⁷ where sss is from 0 to 2 and wherein the solidcarrier is manufactured by binding fully formed peptide nucleic acidprobes to different binding regions on the surface of the solid carrier.2. The solid carrier according to claim 1, wherein said separate anddistinct regions are separated from each other by regions where noprobes are bound.
 3. The solid carrier according to claim 1, whereinsaid probes are all identical in length of bases.
 4. The solid carrieraccording to claim 1, wherein said probes all have a length of between10 and 50 bases.
 5. The solid carrier according to claim 1, wherein thesolid carrier is basically planar.
 6. The solid carrier according toclaim 1, wherein the peptide nucleic acid probes have the generalformula (III):

wherein: each L is independently selected from the group consisting ofhydrogen, phenyl, heterocycles, one, two or three rings, naturallyoccurring nucleobases, and non-naturally occurring nucleobases; each R⁷′is independently selected from the group consisting of hydrogen and theside chains of naturally occurring alpha amino acids; n is an integerfrom 1 to 60; each of k, 1 and m is independently zero or an integerfrom 1 to 5; and optionally the sum of k and m is 1 or 2; R^(h) is OH,NH₂ or —NHLysNH₂; and R^(i) is H or COCH₃.
 7. The solid carrieraccording to claim 1, wherein the peptide nucleic acid probes have abackbone of 2-aminoethyl-glycine subunits.
 8. The solid carrieraccording to claim 1, wherein the binding of the different peptidenucleic acid probes to the different binding regions takes placesimultaneously or sequentially.
 9. The solid carrier according to claim1, wherein reactive groups of the peptide nucleic acid probe andreactive groups of the solid carrier are covalently bound to each otherby means of a linker.
 10. The solid carrier according to claim 9,wherein the linker is more than 15 atoms and less than 200 atoms long.11. A solid carrier comprising at least two single stranded peptidenucleic acid probes of different base sequence covalently bound to thesolid carrier at separate and distinct regions; wherein the probes havethe general formula (I):

wherein: n is at least 2, each of L¹–L^(n) is independently selectedfrom the group consisting of hydrogen, hydroxy, (C₁–C₄) alkanoyl,naturally occurring nucleobases, non-naturally occurring nucleobases,aromatic moieties, DNA intercalators, nucleobase-binding groups andreporter ligands, at least one of L¹–L^(n) being a naturally occurringnucleobase, a non-naturally occurring nucleobase, a DNA intercalator, ora nucleobase-binding group; each of A¹ through A^(n) is a single bond, amethylene group or a group of formula (IIa) or (IIb):

wherein in (IIa) or (IIb): X is O, S, Se, NR³, CH₂ or C(CH₃)₂; Y is asingle bond, O, S or NR⁴; each of p and q is an integer from 1 to 5, thesum p+q being not more than 10; each of r and ss is zero or an integerfrom 1 to 5, the sum r+ss being not more than 10; each R¹ and R² isindependently selected from the group consisting of hydrogen, (C₁–C₄)alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted,hydroxy, alkoxy, alkylthio, amino and halogen; and each R³ and R⁴ isindependently selected from the group consisting of hydrogen,(C₁–C₄)alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C₁–C₄)alkyl,hydroxy, alkoxy, alkylthio and amino; and wherein in formula (1) each ofB¹–B^(n) is N or R³N³⁰, where R³ is as defined above; each of C¹–C^(n)is CR⁶R⁷, CHR⁶CHR⁷ or CR⁶R⁷CH₂, wherein R⁶ is hydrogen and R⁷ isselected from the group consisting of the side chains of naturallyoccurring alpha amino acids, or R⁶ and R⁷ are independently selectedfrom the group consisting of hydrogen, (C₂–C₆) alkyl, aryl, aralkyl,heteroaryl, hydroxy, (C₂–C₆) alkoxy, (C₂–C₆) alkylthio, NR³R⁴ and SR⁵,where R³ and R⁴ are as defined above, and R⁵ is hydrogen, (C₂–C₆)alkyl,hydroxy-, alkoxy-, or alkylthio-substituted (C₂–C₆)alkyl, or R⁶ and R⁷taken together complete an alicyclic or heterocyclic system; each ofD¹–D^(n) is CR⁶R⁷, CH₂CR⁶R⁷ or CHR⁶CHR⁷, where R⁶ and R⁷ are as definedabove; each of G¹–G^(n-1) is

in any orientation, wherein R³ is as defined above; Q is —CO₂H,—CONR′R″, —SO₃H or SO₂NR′R″ or an activated derivative of —CO₂H, —SO₃H;and I is —NHR′″R″″ or —NR′″C(O)R″″, where R′, R″, R′″ and R″″ areindependently selected from the group consisting of hydrogen, alkyl,amino protecting groups, reporter ligands, intercalators, chelators,peptides, proteins, carbohydrates, lipids, steroids, oligonucleotidesand soluble and non-soluble polymers, wherein C and D can optionally beCHR⁶(CH₂)_(sss)CHR⁷ where sss is from 0 to 2 and wherein; reactivegroups of the peptide nucleic acid probe and reactive groups of thesolid carrier are covalently bound to each other by means of a linkerthat is more than 15 atoms and less than 200 atoms long.
 12. The solidcarrier according to claim 11, wherein the linker comprises alkyleneunits.
 13. The solid carrier according to claim 11, wherein the linkercomprises one or more ethylene oxy units and/or peptide groups.
 14. Thesolid carrier according to claim 11, wherein the linker comprises one ormore 8-amino-3,6-dioxa-octanic acid units.
 15. A method for regeneratinga solid carrier in accordance with claim 1 and that has at least twodifferent peptide nucleic acid probes bound to its surface, to which anucleic acid is hybridized, said method comprising: a) treating thecarrier under conditions for denaturing of peptide nucleic acid/nucleicacid complexes; and b) washing the solid carrier to remove denaturednucleic acid polymers.
 16. The method of claim 15, wherein distilledwater at elevated temperature is used to cause denaturing of the peptidenucleic acid/nucleic acid complexes.
 17. The method of claim 15, whereina solution containing sodium hydroxide at elevated temperature is usedto cause denaturing of the peptide nucleic acid/nucleic acid complexes.18. The method of claim 15 wherein the solid carrier is basicallyplanar.
 19. The method of claim 15, wherein the peptide nucleic acidprobes have the general formula (III):

wherein: each L is independently selected from the group consisting ofhydrogen, phenyl, heterocycles, one, two or three rings, naturallyoccurring nucleobases, and non-naturally occurring nucleobases; eachR^(7′) is independently selected from the group consisting of hydrogenand the side chains of naturally occurring alpha amino acids; n is aninteger from 1 to 60; each of k, 1 and m is independently zero or aninteger from 1 to 5; and optionally the sum of k and m is 1 or 2; R^(h)is OH, NH₂ or —NHLysNH₂; and R^(i) is H or COCH₃.
 20. The method ofclaim 15 wherein the peptide nucleic acid probes have a backbone of2-aminoethyl-glycine subunits.